The Molecular Events Of Porcine Reproductive And Respiratory Syndrome Virus Infection Based On Single-particle Tracking Technique

Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)with respiratory and reproductive disorders in pigs.PRRSV has a very restricted cell tropism for cells of the monocytic/macrophage lineage and its infection of the host cells depends on the receptor-mediated endocytosis process.Heparin sulfate,sialoadhesin and CD163 have been identified as the important PRRSV receptors via their involvement in virus attachment,internalization or uncoating in PAMs.Vimentin and Nonmuscle Myosin Heavy Chain II-A(NMHC II-A)are very important for PRRSV infection in Marc-145.Single-particle tracking technique(SPT),a new real-time technology to study the biological dynamic process in situ,which can real-timely monitor virus particle behavior in situ by labeling of viruses and have become an effective means to the study of virus infection mechanism.In order to investigate the behaviors of PRRSV in Marc-145 cells,analyze the effects of vimentin and NMHC II-A on PRRSV infection in Marc-145 cells,and investigate the mechanism of PRRSV infection,this thesis successfully labelled PRRSV with streptavidin-modified quantum dots(SA-QDs)with good stability and strong fluorescence based on the interactions between biotin and streptavidin,and the results were verified by electron microscopy,TCID50,growth curve and immunofluorescence localization.PRRSV particle's individual and group behaviors were long monitored in situ by confocal microscopy.The fusion expression vectors of Green Fluorescent Protein(GFP)with microtubule and microfilament were transfected for labeling of cytoskeleton and studying its role in viral transportation.In order to further explore NMHC II-A and vimentin in PRRSV infection of Marc-145,the expression vectors by fusion of vimentin and NMHC II-A with GFP were constructed.By transient transfection,vimentin and NMHC II-A were labelled in Marc-145 cells for studying their roles during viral infection by Single-Particle Tracking Technique.It was resulted that:(i)Successful labeling of PRRSV with quantum dots showed that the labeling method had little effect on the biological activity of virus particles,and the labeling efficiency was high;(ii)In Marc-145 cells,PRRSV moves from the periphery of cells to the nucleus via slow-fast-slow intermittent movements,most of which converge to specific regions;(iii)PRRSV transports to the nucleus relying on microtubules and microfilaments,and most of which gather to a certain area;(iv)Some viruses move from the periphery of the cell to the nucleus around vimentin,and vimentin may be involved in the intracellular transportation of PRRSV;(v)NMHC II-A has obvious colocalization with PRRSV,which may be related to intracellular trafficking of virus PRRSV.The application of SPT in PRRSV infection mechanism provides a new technical means for PRRSV research,which is helpful to promote the research of PRRSV infection mechanism.The research of vimentin and NMHC II-A is helpful to reveal PRRSV infection mechanism and lays a solid foundation for the development of more effective viral vaccines and diagnostic reagents.